Due to limitations associated with autografted bone, allograft, xenograft and alloplast materials are commonly used for bone grafting procedures. However these materials lack osteoinductivity. The proposed research aims to couple osteoinductive factors to the surface of these alternative graft sources in order to enhance osteoregeneration in sites of bone deficiency. Our group is utilizing a unique approach involving the addition of a calcium-binding amino acid domain, heptaglutamate (E7), to an osteoinductive peptide derived from BMP2. Preclinical studies show that graft materials coupled with the E7-modified BMP2 peptide (E7-BMP2pep) stimulated significantly greater new bone synthesis than uncoated materials in both a subcutaneous (ectopic) and mandibular defect model. Importantly, bone formation was comparable to, and in some cases greater than, that induced by graft coated with full-length rBMP2, and no side effects were noted for the E7-BMP2pep group. In contrast, animals implanted with rBMP2 developed chronic inflammation and cataracts. We hypothesize that the E7-BMP2pep is an effective, safe, and readily translatable agent that can be used to enhance the performance of a wide array of bone grafting materials including allograft, xenograft and alloplast. There are two major objectives of this proposal: (1) to comprehensively analyze host response to the E7- BMP2pep in order to advance the research toward translation (Aim 1); and (2) to elucidate the mechanism by which BMP2 mimetic peptides activate osteoprogenitor cells, with the broader goal of identifying a BMP2 derivative with maximal osteoinductive potential. AIM 1: Host response to E7-BMP2pep-coupled graft materials A mandibular defect model will be used to assess the effects of E7-BMP2pep coupled materials on immune and foreign body response, fibrosis, angiogenesis, quantity and quality of newly-formed bone, and implant integration. Dissemination of the peptides and rBMP2 away from the implant site will also be monitored. We hypothesize that E7-BMP2pep will remain tightly localized within the defect, promoting rapid and effective bone healing. AIM 2: Molecular characterization and engineering of BMP2 mimetics to enhance osteoinductive potential Using cell culture and calvarial organ culture systems, the mechanisms by which E7-modified BMP2 mimetic peptides regulate stem/progenitor cells will be elucidated. Additionally, BMP2 peptide variants with activating mutations will be developed, and in complementary studies, an E7 domain will be engineered onto full-length rBMP2 to improve coupling to carriers. These BMP variants will be evaluated for receptor binding activity and induction of cell signaling and osteoblastogenesis. Enhanced osteoinductivity of the variants will be confirmed by mandibular implant studies. The broad goal of Aim 2 is to identify the ideal BMP2 cargo molecule for E7- directed coupling to graft materials.